The PI is a clinically trained neurologist specializing in Parkinson's disease (PD). The PI has been studying the molecular mechanism of I -methyl-4-phenyl- 1,2,3,6- tetrahydropyridine (MPTP), a toxin that damages substantia nigra (SN) dopamine (DA) neurons as seen in PD. The PI's work has provided compelling evidence that both superoxide radicals and nitric oxide (NO) are implicated in MPTP's toxicity. Both species are modestly reactive, but can combine to produce the highly reactive tissue-damaging peroxynitrite. To elucidate the source of N0 that participates in MPTP's toxicity, Specific Aim (SA)-I will compare the effects of MPTP on SN DA neurons of mutant mice deficient in neuronal, inducible, or endothelial NO synthase (NOS), the three isoforms of NOS. To demonstrate the production of peroxynitrite following MPTP administration, SA-II will quantify striatal and midbrain levels of nitrotyrosine, a stable fingerprint of peroxynitrite's deleterious effects on proteins, at different time-points and doses of MPTP. The requirement for superoxide, NO, and MPTP's active metabolite, 1- methyl-4-phenylpyridinium in protein nitrotyrosine formation, as well as its specificity for DA neurons will also be examined. To define the cell groups and organelles preferentially tyrosine-nitrated, SA-III will ascertain the cellular and subcellular distribution of nitrotyrosine immunoreactivity in the mouse midbrain after MPTP administration. To examine the potential biological consequences of protein tyrosine nitration, SA-IV will assess whether candidate proteins, tyrosine hydroxylase and manganese superoxide dismutase, are nitrated after MPTP administration. The catalytic activity of these enzymes and the search for other proteins that are tyrosine-nitrated after MPTP administration will also be undertaken. This proposal contains a comprehensive set of experiments, which should provide important insights into the role of NO in MPTP toxicity. It should also shed light on the mechanism(s) of neurodegeneration in PD.